A central question in the study of T cell mediated autoimmune diseases remains the identification of the cognate self-target(s) recognized by pathogenic T lymphocytes and the cellular regulation of these autoimmune responses. In patients with Type 1 diabetes, studies have been limited to peripheral blood. However, it is known that self-antigens are transported specifically to pancreatic lymph nodes (PLN) and trigger autoreactive T cell activation. Regulatory T cell populations at the site of antigen presentation in the draining lymph nodes are thought to influence and direct the generation and phenotype of auto-reactive cells. We have developed techniques to examine pancreatic lymph node T cells from patients with diabetes and control subjects using single cell cloning. Our aims are to determine if there is oligoclonal expansion of PLN T cells in patients with diabetes, to determine antigen-specificity of clonally expanded T cell clones isolated from patients by screening a human islet-specific cDNA library and combinatorial peptide library, and to determine if the phenotype and frequency of NKT cells derived from PLN of patients with diabetes is altered. Our preliminary data demonstrate that there is clonal expansion of T cells in the PLN of patients with diabetes but not controls. By comparing CDR3 regions of the TCRalpha and TCRbeta chains from clones, we observed that in each patient sample, almost half of the total T cell clones isolated express the same Vbeta chain. No obvious clonal expansion was observed in the control subjects. We have constructed a human pancreatic islet cell-specific cDNA library that will be used to transfect autologous EBV transformed B cells and these cells will be used as antigen presenting cells to hybridomas transfected with the TCRalpha and beta chains and an IL-2 responsive reporter gene. We will use a combinatorial peptide library based on DRB1*0401 as another source of stimulating peptides. A unique sample is from a new onset diabetic from which we have observed lymphocytic infiltrate containing CD4+ T cells in pancreatic islets. We will laser microcapture individual T cells from islets and identify TCRalpha and beta chain usage. This will give us direct evidence of antigen reactivity of an islet-infiltrating T cell in human Type 1 diabetes. We single cell cloned NKT cells from the PLN by Valpha24 antibody onto mitogen-activated irradiated, allogeneic feeders, by Valpha24 antibody onto to alpha-galactosylceramide pulsed feeders, and by sorting with a CD1d tetramer and measured their frequency and function. We have found that NKT cells derived from normal PLN secrete both IL-4 and gammaIFN, which is the normal phenotype of these cells. However, the NKT cells derived from diabetic PLN were unable to secrete IL-4. NKT cells derived from the new onset PLN are a mixture of cells with the normal phenotype and loss of IL-4 secretion phenotype. Thus, our goal is to identify the self-antigen driving the clonal expansion of T cells in the PLN of patients with autoimmune diabetes, and determine if this clonal expansion is associated with dysfunction of regulatory NKT cells. These investigations are designed to provide novel targets and approaches for the treatment of human autoimmune disease.